Graft prosthesis, material and methods

ABSTRACT

A graft prostheses ( 11 ), materials and method for implanting, transplanting, replacing, or repairing a part of a patient. The graft prosthesis includes a purified, collagen-based matrix structure removed from a submucosa tissue source. The submucosa tissue source is purified by disinfection and removal steps to deactivate and remove contaminants, thereby making the purified structure biocompatible and suitable for grafting on and/or in a patient.

REFERENCE TO RELATED APPLICATIONS

This application claims priority upon U.S. Patent Application Ser. Nos.60/024,542 and 60/024,693, filed on Aug. 23, 1996 and Sep. 6, 1996,respectively, each of which is hereby incorporated by reference in itsentirety.

TECHNICAL FIELD

This invention relates generally to a medical structure and, inparticular, to a graft prosthesis, materials, and methods therefor.

BACKGROUND OF THE INVENTION

Tissue implants in a purified form and derived from collagen-basedmaterials have been manufactured and disclosed in the literature.Cohesive films of high tensile strength have been manufactured usingcollagen molecules or collagen-based materials. Aldehydes, however, havebeen generally utilized to cross-link the collagen molecules to producefilms having high tensile strengths. With these types of materials, thealdehydes may leech out of the film, e.g. upon hydrolysis. Because suchresidues are cytotoxic, the films are poor tissue implants.

Other techniques have been developed to produce collagen-based tissueimplants while avoiding the problems associated with aldehydecross-linked collagen molecules. One such technique is illustrated inU.S. Pat. No. 5,141,747 wherein the collagen molecules are cross-linkedor coupled at their lysine epsilon amino groups followed by denaturingthe coupled, and preferably modified, collagen molecules. The discloseduse of such collagen material is for tympanic membrane repair. Whilesuch membranes are disclosed to exhibit good physical properties and tobe sterilized by subsequent processing, they are not capable ofremodeling or generating cell growth or, in general, of promotingregrowth and healing of damaged or diseased tissue structures.

In general, researchers in the surgical arts have been working for manyyears to develop new techniques and materials for use as implants andgrafts to replace or repair damaged or diseased tissue structures, forexample, blood vessels, muscle, ligaments, tendons and the like. It isnot uncommon today, for instance, for an orthopedic surgeon to harvest apatellar tendon of autogenous or allogenous origin for use as areplacement for a torn cruciate ligament. The surgical methods for suchtechniques are known. Further, it has been common for surgeons to useimplantable prostheses formed from plastic, metal and/or ceramicmaterial for reconstruction or replacement of physiological structures.Yet, despite their wide use, surgical implanted prostheses present manyattendant risks to the patient.

Researchers have also been attempting to develop satisfactory polymer orplastic materials to serve as such functional tissue structures and/orother connective tissues, e.g., those involved in hernia and jointdislocation injuries. It has been discovered that it is difficult toprovide a tough, durable plastic material which is suitable for longterm connective tissue replacement. The tissues surrounding the plasticmaterial can become infected and difficulties in treating suchinfections often lead to the failure of the implant or prostheses.

As mentioned above, various collagen-based materials have also beenutilized for the above-mentioned tissue replacements; however, thesematerials either did not exhibit the requisite tensile strength or alsohad problems with infection and other immunogenic responses,encapsulation, or had other problems when they may have been loaded withantibiotics, growth factors and the like. For example, U.S. Pat. No.4,956,178 discloses a submucosa collagen matrix which is obtained fromthe intestinal tract of mammals; however, it is disclosed that thecollagen matrix is loaded with antibiotics. In a related patent, U.S.Pat. No. 5,372,821, it is disclosed that a submucosa collagen matrix maybe sterilized by conventional techniques, e.g., aldehyde tanning,propylene oxide, gamma radiation and peracetic acid. No specificprocessing steps are disclosed except that the submucosa layer is firstdelaminated from the surrounding tissue prior to sterilizationtreatment.

Therefore, there is a need to obtain improved purified forms ofcollagen-based matrices from tissue sources thereof. Also, there is aneed to provide a process whereby the ease of removal of such matricesfrom tissue sources is enhanced so as to yield such improved, purifiedproducts. The present invention is addressed to these needs.

SUMMARY OF THE INVENTION

In accordance with one preferred embodiment of the present invention,provided is a graft prosthesis which includes a purified, collagen-basedmatrix structure removed from a submucosa tissue source, wherein thepurified structure has a contaminant level making the purified structurebiocompatible.

Another preferred embodiment of the invention provides a graftprosthesis which includes a purified, collagen-based matrix structureremoved from a submucosa tissue source, wherein the purified structurehas an endotoxin level of less than 12 endotoxin units per gram.

Another preferred embodiment of the invention provides a graftprosthesis which includes a purified, collagen-based matrix structureremoved from a submucosa tissue source, wherein the purified structurehas a nucleic acid content level of less than 2 micrograms permilligram.

Another preferred embodiment of the invention provides a graftprosthesis including a purified, collagen-based matrix structure removedfrom a submucosa tissue source, wherein the purified structure has avirus level of less than 500 plaque forming units per gram.

The present invention also provides a graft prosthesis which includes apurified, collagen-based matrix structure removed from a submucosatissue source, wherein the purified structure has a processing agentlevel of less than 100,000 parts per million per kilogram.

A further embodiment of the invention concerns a method for obtaining acollagen-based matrix from a submucosa tissue source. The methodincludes treating the submucosa tissue source with a disinfecting agentto provide a disinfected submucosa tissue source, and removing thecollagen-based matrix from the disinfected submucosa tissue source.

Another preferred embodiment of the invention provides a method forobtaining a collagen-based matrix from a submucosa tissue source, whichincludes providing a submucosa tissue source which has been treated witha disinfecting agent, and removing the collagen-based matrix from thesubmucosa tissue source.

The present invention also concerns a composition which includes acollagen-containing structure removed from a tissue source initiallycontaining the structure and other tissue, wherein thecollagen-containing structure has an endotoxin level of no greater than12 endotoxin units per gram.

Also provided by the present invention is a purified collagen-containingmatrix obtained from a mammalian tissue source, the matrix includingmammalian tela submucosa and being obtainable by a process whichincludes disinfecting the mammalian tissue source then removing thestructure from the resulting disinfected mammalian tissue source.

In preferred aspects, the invention provides purified forms of telasubmucosa collagen matrices derived from the alimentary, respiratory,urinary or genital tracts of animals, the matrices having a bioburdenlevel of substantially zero, and/or being essentially free of pyrogens.A preferred collagen matrix is capable of being implanted within a humanor animal patient without causing a cytotoxic response, infection,rejection of the implant or any other harmful effect in a majority ofpatients. While a preferred implantable collagen matrix according tosome aspects of the present invention comprises primarily telasubmucosa, the collagen matrix in this instance may also comprisepartial layers of laminar muscularis mucosa, muscularis mucosa, laminapropria, a stratum compactum layer and/or other such tissue materialsdepending upon the source from which it was derived.

Further in accordance with the present invention, a purified delaminatedtela submucosa collagen matrix is provided which is derived from thealimentary, respiratory, urinary or genital tracts of animals or humans,wherein said purified submucosa collagen matrix is produced bydelaminating a disinfected tela submucosa source to obtain thedelaminated tela submucosa collagen matrix. An advantageous matrix maybe obtained, for example, by a process comprising treating anunprocessed, undelaminated tela submucosa source harvested from thealimentary, respiratory, urinary or genital tracts of animals with adisinfecting agent, followed by delaminating the tela submucosa collagenmatrix from the attached tissues. The preferred collagen matrix has abioburden level of substantially zero and capable of being implantedwithin a human or animal patient without causing a cytotoxic response,infection, rejection of the implant or any other harmful effect in amajority of patients.

Still further in accordance with the present invention, a method isprovided for obtaining a highly pure, delaminated tela submucosacollagen matrix in a substantially sterile state, comprisingdelaminating a disinfected tela submucosa tissue source to obtain thedelaminated tela submucosa collagen matrix. A preferred method comprisestreating an undelaminated tela submucosa source harvested from thealimentary, respiratory, urinary or genital tracts of animals or humanswith a disinfecting agent, followed by delaminating the tela submucosafrom its other source tissues attached to the tela submucosa.

Still further in accordance with the present invention, provided is ahighly pure tela submucosa collagen matrix derived from the alimentary,respiratory, urinary or genital tracts of animals having a bioburden ofsubstantially zero, and wherein said tela submucosa collagen matrixcontains substantially no surface debris, e.g. including substantiallyno muscle tissue, mucosal layers, lipids or cellular debris. Thepreferred collagen matrix is capable of being implanted within a humanor animal patient without causing cytotoxic response, infection,rejection of the implant or any other harmful effect to the patient.

Still further in accordance with the present invention, a highly puretela submucosa is provided which is derived from the alimentary,respiratory, urinary or genital tracts of animals and wherein the telasubmucosa is delaminated in a substantially sterile condition comprisinggrowth factors, and is produced by rinsing the delaminated, telasubmucosa source with a solvent, for instance water, followed bytreatment with a disinfecting agent, preferably a peracid, at a pH ofabout 1.5 to about 10 followed by delamination of the tela submucosafrom the attached tissues. The peracid is buffered at pH levels greaterthan 7. Desirably, collagen matrices so produced have a substantial highcontent of one or more growth factors.

Still further in accordance with the present invention, provided is atissue graft composition which includes a tela submucosa collagen matrixwhich is essentially pyrogen free. More preferred such compositions willinclude a tela submucosa collagen matrix which has a pyrogen content ofabout 1 endotoxin unit per gram (EU/g) or less.

Still further in accordance with the present invention, a highly pure,tela submucosa as described above, will demonstrate active angiogenesisin vivo upon implantation in a human or animal patient.

This invention relates to purified, implantable tissue constructs, aprocess for producing such purified, implantable tissue constructs, andtheir use to promote regrowth and healing of damaged or diseased tissuestructures. More particularly, the invention is directed to purifiedforms of tela submucosa collagen matrix suitable for use as animplantable tissue, and methods for producing such purified forms ofthis collagen-based implantable tissue.

These and other aspects of the present invention will become apparent tothose skilled in the art upon reviewing the specification that follows.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides a perspective view of a tubular graft prosthesisstructure in accordance with the invention.

DETAILED DESCRIPTION

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to certain preferred embodimentsthereof and specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations, further modificationsand applications of the principles of the invention as described hereinbeing contemplated as would normally occur to one skilled in the art towhich the invention relates.

In the discussions herein, a number of terms are used. In order toprovide and clear and consistent understanding of the specification andclaims, the following definitions are provided.

Bioburden—refers to the number of living microorganisms, reported incolony-forming units (CFU), found on and/or in a given amount ofmaterial. Illustrative microorganisms include bacteria, fungi and theirspores.

Disinfection—refers to a reduction in the bioburden of a material.

Sterile—refers to a condition wherein a material has a bioburden suchthat the probability of having one living microorganism (CFU) on and/orin a given section of the material is one in one-million or less.

Pyrogen—refers to a substance which produces febrile response afterintroduction into a host.

Endotoxin—refers to a particular pyrogen which is part of the cell wallof gram-negative bacteria. Endotoxins are continually shed from thebacteria and contaminate materials.

Purification—refers to the treatment of a material to remove one or morecontaminants which occur with the material, for instance contaminantswith which the material occurs in nature, and/or microorganisms orcomponents thereof occurring on the material. Illustratively, thecontaminants may be those known to cause toxicity, infectivity,pyrogenicity, irritation potential, reactivity, hemolytic activity,carcinogenicity and/or immunogenicity.

Biocompatibility—refers to the ability of a material to pass thebiocompatibility tests set forth in International Standards Organization(ISO) Standard No. 10993 and/or the U.S. Pharmacopeia (USP) 23 and/orthe U.S. Food and Drug Administration (FDA) blue book memorandum No.G95-1, entitled “Use of International Standard ISO-10993, BiologicalEvaluation of Medical Devices Part-1: Evaluation and Testing.”Typically, these tests assay as to a material's toxicity, infectivity,pyrogenicity, irritation potential, reactivity, hemolytic activity,carcinogenicity and/or immunogenicity. A biocompatible structure ormaterial when introduced into a majority of patients will not cause anadverse reaction or response. In addition, it is contemplated thatbiocompatibility can be effected by other contaminants such as prions,surfactants, oligonucleotides, and other biocompatibility effectingagents or contaminants.

Contaminant—refers to an unwanted substance on, attached to, or within amaterial. This includes, but is not limited to: bioburden, endotoxins,processing agents such as antimicrobial agents, blood, blood components,viruses, DNA, RNA, spores, fragments of unwanted tissue layers, cellulardebris, and mucosa.

Tela submucosa—refers to a layer of collagen-containing connectivetissue occurring under the mucosa in most parts of the alimentary,respiratory, urinary and genital tracts of animals.

As disclosed above, the present invention generally provides graftprostheses and materials including a purified collagen-based matrixstructure, and methods for obtaining and using the same. Advantageousgraft prostheses of the invention are obtained from a submucosa tissuesource, for example including animal tissues such as human or othermammalian tissues, e.g. porcine, bovine or ovine tissues.

Tela submucosa, as with many animal tissues, is generally aseptic in itsnatural state, provided the human or animal does not have an infectionor disease. This is particularly the case since the tela submucosa is aninternal layer within the alimentary, respiratory, urinary and genitaltracts of animals. Accordingly, it is generally not exposed to bacteriaand other cellular debris such as the epithelium of the intestinaltract. One feature of the present invention is the discovery that bydisinfecting the source tissue for the tela submucosa prior todelamination, the aseptic state of the tela submucosa layer can bepreserved or substantially preserved, particularly if the delaminationprocess occurs under sterile conditions.

In particular, it has been discovered that disinfecting the telasubmucosa source, followed by removal of a purified matrix including thetela submucosa, e.g. by delaminating the tela submucosa from the tunicamuscularis and the tunica mucosa, minimizes the exposure of the telasubmucosa to bacteria and other contaminants. In turn, this enablesminimizing exposure of the isolated tela submucosa matrix todisinfectants or sterilants if desired, thus substantially preservingthe inherent biochemistry of the tela submucosa and many of the telasubmucosa's beneficial effects.

A tela submucosa implantable collagen matrix according to the presentinvention can, as indicated above, be obtained from the alimentary,respiratory, urinary or genital tracts of animals. Preferably, the telasubmucosa tissues, which are collagen-based and thus predominantlycollagen, are derived from the alimentary tract of mammals and mostpreferably from the intestinal tract of pigs. A most preferred source ofwhole small intestine is harvested from mature adult pigs weighinggreater than about 450 pounds. Intestines harvested from healthy,nondiseased animals will contain blood vessels and blood supply withinthe intestinal tract, as well as various microbes such as E. colicontained within the lumen of the intestines. Therefore, disinfectingthe whole intestine prior to delamination of the tela submucosasubstantially removes these contaminants and provides a preferredimplantable tela submucosa tissue which is substantially free of bloodand blood components as well as any other microbial organisms, pyrogensor other pathogens that may be present. In effect, this procedure isbelieved to substantially preserve the inherent aseptic state of thetela submucosa, although it should be understood that it is not intendedthat the present invention be limited by any theory.

It is also desirable that the collagen matrix according to the presentinvention be substantially free of any antibiotics, antiviral agents orany antimicrobial type agents which may affect the inherent biochemistryof the matrix and its efficacy upon implantation. In the past, onemethod of treating such tissue material is to rinse the delaminatedtissue in saline and soak it in an antimicrobial agent, for example, asdisclosed in U.S. Pat. No. 4,956,178. While such techniques canoptionally be practiced with isolated submucosa of the presentinvention, preferred processes according to present invention avoid theuse of antimicrobial agents and the like which may not only affect thebiochemistry of the collagen matrix but also can be unnecessarilyintroduced into the tissues of the patient.

As discussed above, it has been discovered that a highly pure form of animplantable tela submucosa collagen matrix may be obtained by firstdisinfecting a tela submucosa source prior to removing a purifiedcollagen matrix including the tela submucosa layer, e.g. by delaminatingthe tela submucosa source. It has also been discovered that certainprocessing advantages as well as improved properties of the resultanttela submucosa layer are obtained by this process, including greaterease in removing attached tissues from the submucosa layer, and acharacteristic, low contaminant profile.

Processes of the invention desirably involve first rinsing the telasubmucosa source one or more times with a solvent, suitably water. Therinsing step is followed by treatment with a disinfecting agent. Thedisinfecting agent is desirably an oxidizing agent. Preferreddisinfecting agents are peroxy compounds, preferably organic peroxycompounds, and more preferably peracids. Such disinfecting agents aredesirably used in a liquid medium, preferably a solution, having a pH ofabout 1.5 to about 10, more preferably a pH of about 2 to about 6, andmost preferably a pH of about 2 to about 4. In methods of the presentinvention, the disinfecting agent will generally be used underconditions and for a period of time which provide the recovery ofcharacteristic, purified submucosa matrices as described herein,preferably exhibiting a bioburden of essentially zero and/or essentialfreedom from pyrogens. In this regard, desirable processes of theinvention involve immersing the tissue source (e.g. by submersing orshowering) in a liquid medium containing the disinfecting agent for aperiod of at least about 5 minutes, typically in the range of about 5minutes to about 40 hours, and more typically in the range of about 0.5hours to about 5 hours.

A preferred peroxy disinfecting agent is hydrogen peroxide. Theconcentration of hydrogen peroxide can range from about 0.05% to 30% byvolume. More preferably the hydrogen peroxide concentration is fromabout 1% to 10% by volume and most preferably from about 2% to 5% byvolume. The solution may or may not be buffered to a pH from about 5 to9. More preferably the pH is from about 6 to 7.5. These concentrationscan be diluted in water or in an aqueous solution of about 2% to about30% by volume alcohol. Most preferably the alcohol is ethanol. Thesolution temperature can range from about 15 to 50° C. More preferablythe solution temperature is from about 20 to 40° C. Most preferably, thesolution temperature is from about 32 to 37° C. The exposure time canrange from about 10 to 400 minutes. Preferably, the exposure time isfrom about 120 to 240 minutes. More preferably, the exposure time isfrom 180 to 210 minutes.

A preferred organic peroxide disinfecting agent is perpropionic acid.The concentration of perpropionic acid may range from about 0.1% to 10%by volume. More preferably the perpropionic acid concentration is fromabout 0.1% to 1.0% by volume and most preferably from about 0.2% to 0.5%by volume. These concentrations of perpropionic acid can be diluted inwater or in an aqueous solution of about 2% to about 30% by volumealcohol. Most preferably the alcohol is ethanol. The tela submucosatissue source can be exposed to the organic peroxide solution forperiods from about 15 minutes to about 40 hours, and more typically inthe range of about 0.5 hours to about 8 hours. Other peroxy disinfectingagents are suitable for use as described in “Peroxygen Compounds”, S.Block, in Disinfection, Sterilization and Preservation, S. Block,Editor, 4th Edition, Philadelphia, Lea & Febiger, pp. 167-181, 1991; and“Disinfection with peroxygens”, M. G. C. Baldry and J. A. L. Fraser, inIndustrial Biocides, K. Payne, Editor, New York, John Wiley and Sons,pp. 91-116, 1988.

Another oxidizing disinfecting agent is chlorhexidine(1,6-di(4-chlorophenyldiguanido)hexane) in its digluconate form. Theconcentration of chlorhexidine digluconate may range from about 0.1% to15% by weight. More preferably, the chlorhexidine digluconateconcentration is from about 0.1% to 2% by weight and most preferablyfrom about 0.2% to 5% by weight. The solution may or may not be bufferedto a pH from about 5 to 8. More preferably the pH is from about 5.5 to7. These concentrations may be diluted in water or in an aqueoussolution of about 2% to about 20% by volume alcohol. Most preferably thealcohol is ethanol at a concentration of about 5% to 10%. The solutiontemperature may range from about 15 to 30° C. The exposure time mayrange from about 10 to 400 minutes. More preferably the exposure time isfrom about 30 to 60 minutes. Other chlorine agents are described in“Chlorhexidine”, G. W. Denton, in Disinfection, Sterilization andPreservation, S. Block, Editor, 4th Edition, Philadelphia, Lea &Febiger, pp. 274-289, 1991.

In preferred preparative processes, a peracid or other disinfectingagent may be dissolved in a dilute aqueous alcohol solution, preferablywherein the alcohol has from 1 to about 6 carbon atoms, and wherein thealcohol may generally comprise from about 1% to about 30% by volume ofthe solution. More preferred alcohols for use in the invention areselected from the group consisting of ethanol, propanols and butanols.Ethanol is a most preferred alcohol for these purposes.

When a peracid is used in the disinfection, it is preferably selectedfrom the group consisting of peracetic acid, perpropionic acid orperbenzoic acid. Peracetic acid is the most preferred disinfectingagent. The peracetic acid is preferably diluted into about a 2% to about10% by volume alcohol solution. The concentration of the peracetic acidmay range, for example, from about 0.05% by volume to about 1.0% byvolume. Most preferably the concentration of the peracetic acid is fromabout 0.1% to about 0.3% by volume. Hydrogen peroxide can also be usedas a disinfecting agent. Alternatively, or in addition, the telasubmucosa tissue source, e.g. from small intestine, may be disinfectedutilizing disinfecting agents such as glutaraldehyde, formalin and thelike, which are also known for their ability to introduce substantialcrosslinking into collagen matrices, in contrast to the action of otherdisinfecting agents such as peracids which can be used to disinfectwithout introducing such crosslinking. Additionally, the tela submucosasource can be treated with radiation, e.g., gamma radiation, forpurposes of disinfection.

Variations on the disinfection process can also include the following:

-   1. Intestine is treated with 0.2% peracetic acid, 5% ethanol    solution at a ratio of 10:1 solution to intestine ratio by weight.    Solution has a pH of 2.6. Solution and intestine are vigorously    mixed for two hours.-   2. Intestine is treated with 1% peracetic acid, 25% ethanol solution    at a ration of 5:1 solution to intestine ratio by weight. Solution    has a pH of 2. Solution and intestine are vigorously mixed for one    hour.-   3. Intestine is treated with 1% peracetic acid, 15% ethanol, and 10%    hydrogen peroxide solution at a ratio of 5:1 solution to intestine    ratio by weight. Solution and intestine are vigorously mixed for one    hour.-   4. Whole small intestine is rinsed four times with high purity water    for 15 minutes. The intestine is then subjected to 1.5 MRAD Electron    Beam radiation.-   5. Whole small intestine is rinsed four times with high purity water    for 15 minutes. Lengthwise along a conveyor belt, the intestine is    subjected to high-intensity pulsed light which disinfects the    intestine.

Following the treatment as described above, the tela submucosa layer isdelaminated from its source, e.g., whole intestine, cow uterus and thelike. It has been found that by following thispost-disinfection-stripping procedure, it is easier to separate the telasubmucosa layer from the attached tissues, e.g. at least from attachedtunica muscularis tissue, as compared to stripping the tela submucosalayer prior to disinfection. Moreover it has been discovered that theresultant tela submucosa layer in its most preferred form exhibitssuperior histology, in that there is less attached tissue and debris onthe surface compared to a tela submucosa layer obtained by firstdelaminating the tela submucosa layer from its source and thendisinfecting the layer. Moreover, a more uniform tela submucosa tissuecan be obtained from this process, and a tela submucosa having the sameor similar physical and biochemical properties can be obtained moreconsistently from each separate processing run. Importantly, a highlypurified, substantially sterile tela submucosa is obtained by thisprocess.

The stripping of the tela submucosa source is preferably carried out byutilizing a disinfected or sterile casing machine, to produce a telasubmucosa which is substantially sterile and which has been minimallyprocessed. A suitable casing machine is the Model 3-U-400 StridhsUniversal Machine for Hog Casing, commercially available from the ABStridhs Maskiner, Gotoborg, Sweden. Therefore, the measured bioburdenlevels are minimal or substantially zero. Of course, other means fordelaminating the tela submucosa source can be employed without departingfrom the present invention, including for example delaminating by hand.

It has also been discovered that more preferred processes according tothe present invention, not only will eliminate or significantly reducecontaminants contained in the tela submucosa collagen matrix, but alsowill produce a tissue which exhibits no substantial degradation ofphysical and mechanical properties, e.g., differential porosity (i.e.wherein one side of the submucosa layer has greater porosity than theother side), and good strength, for example burst strength. Also, it hasbeen discovered that more preferred processes do not affect thedifferential porosity of the tela submucosa collagen matrix whichultimately affects the level of efficacy of this tissue implant. Forexample, the tissue is not necessarily treated with a crosslinking agentor a material that disrupts the porosity or inherent, native structureof the collagen matrix. Moreover, when hydrogen peroxide is employed,the matrix as a whole has greater porosity as well as a higher oxygencontent. This helps to ensure the absence of contaminants e.g.,endotoxins, pyrogens and the like.

Also, in an advantageous form, the collagen-based matrices of thepresent invention (e.g., including tela submucosa) demonstrate theability to induce active angiogenesis, i.e., an ingrowth of bloodvessels within the matrix of the tissue. In this regard, these preferredmatrices of the invention will contain beneficial components with whichthe matrices naturally occur, including for example one or more ofglycosaminoglycans, glycoproteins, proteoglycans, and/or growth factors(e.g. Transforming Growth Factor-á, Transforming Growth Factor-â, and/orFibroblast Growth Factor 2 (basic)).

Preferred collagen-based matrices of the invention, preferablysubmucosa-containing matrices, are also characterized by the lowcontaminant levels set forth in Table 1 below, each contaminant leveltaken individually or in any combination with some or all of the otherdisclosed contaminant levels. The abbreviations in Table 1 are asfollows: CFU/g=colony forming units per gram; PFU/g=plaque forming unitsper gram; ig/mg=micrograms per milligram; ppm/kg=parts per million perkilogram.

TABLE 1 FIRST SECOND THIRD PREFERRED PREFERRED PREFERRED FEATURE LEVELLEVEL LEVEL ENDOTOXIN <12 EU/g <10 EU/g <5 EU/g BIOBURDEN <2 CFU/g <1CFU/g <0.5 CFU/g FUNGUS <2 CFU/g <1 CFU/g <0.5 CFU/g NUCLEIC ACID <10μg/mg <5 μg/mg <2 μg/mg VIRUS <500 PFU/g <50 PFU/g <5 PFU/g PROCESSING<100,000 <1,000 <100 AGENT ppm/kg ppm/kg ppm · kg

Even more preferred collagen-based matrices of the invention contain anendotoxin level of less than 1 EU/g, and most preferably less than 0.5EU/g.

Purified collagen-based matrices according to the present invention maybe processed in a number of ways, to provide collagenous matrices usefulboth in vitro and in vivo. For example, the submucosa may be configuredto provide tissue grafts useful in vascular applications, e.g. asgenerally described in U.S. Pat. Nos. 2,127,903 and 4,902,508. Withreference now to FIG. 1, for use in vascular grafting, a generallytubular graft prosthesis structure 11 is formed with or including thecollagen-based matrix 12, the diameter “D” of which approximates that ofa recipient blood vessel. In one mode, this may be accomplished bymanipulating a tubular segment or sheet of the tela submucosa to definea cylinder having a diameter “D” approximately the same as that of therecipient blood vessel, and suturing, bonding or otherwise securing thelongitudinal seam 13 to form an appropriately-dimensioned tubularvascular graft having a lumen 14 for passage of blood. In illustrativepreparative procedures, the graft is formed over a sterile rod ormandrel having an outer diameter approximately equal to that of thevessel to be grafted. For instance, the rod is introduced into the lumenof a tela submucosa segment retaining its native, tubular form.Redundant tissue is then gathered, and the desired lumen diameterachieved by suturing along the length of the graft (for example, usingtwo continuous suture lines or a simple, interrupted suture line), or byusing other art-recognized tissue securing techniques. Alternatively, asheet of the inventive tela submucosa is wrapped about the rod to forman overlapping seam, which can be sutured, glued or otherwise secured,to provide the tubular graft construct. In preferred forms, the inner,luminal surface of the graft can be formed by the mucosal side of thetela submucosa.

The tela submucosa of the invention possesses mechanical propertieshighly desirable for tissue graft materials in vascular applications,including low porosity index, high compliance, and a high burststrength. One skilled in the art will appreciate that the preferredtissue graft material will be of low enough porosity to preventintraoperative hemorrhage and yet of high enough porosity to allowextension of a newly-developed vasa vasorum through the graft materialto nourish the neointima and luminal surface.

Tela submucosa tissue of the present invention can also be processed toprovide fluidized compositions, for instance using techniques asdescribed in U.S. Pat. No. 5,275,826. In this regard, solutions orsuspensions of the tela submucosa can be prepared by comminuting and/ordigesting the tela submucosa with a protease (e.g. trypsin or pepsin),for a period of time sufficient to solubilize the tissue and formsubstantially homogeneous solution. The submucosa starting material isdesirably comminuted by tearing, cutting, grinding, shearing or thelike. Grinding the submucosa in a frozen or freeze-dried state isadvantageous, although good results can be obtained as well bysubjecting a suspension of pieces of the submucosa to treatment in ahigh speed blender and dewatering, if necessary, by centrifuging anddecanting excess waste. The comminuted tela submucosa can be dried, forexample freeze dried, to form a powder. Thereafter, if desired, thepowder can be hydrated, that is, combined with water or buffered salineand optionally other pharmaceutically acceptable excipients, to form afluid tissue graft composition, e.g. having a viscosity of about 2 toabout 300,000 cps at 25EC. The higher viscosity graft compositions canhave a gel or paste consistency.

Fluidized tela submucosa of this invention finds use as an injectableheterograft for tissues, for example, bone or soft tissues, in need ofrepair or augmentation most typically to correct trauma ordisease-induced tissue defects. The present fluidized submucosacompositions are also used advantageously as a filler for implantconstructs comprising, for example, one or more sheets of tela submucosaformed into sealed (sutured) pouches for use in cosmetic ortrauma-treating surgical procedures.

In one illustrative preparation, tela submucosa prepared as describedherein is reduced to small pieces (e.g. by cutting) which are charged toa flat bottom stainless steel container. Liquid nitrogen is introducedinto the container to freeze the specimens, which are then comminutedwhile in the frozen state to form a coarse tela submucosa powder. Suchprocessing can be carried out, for example, with a manual arbor presswith a cylindrical brass ingot placed on top of the frozen specimens.The ingot serves as an interface between the specimens and the arbor ofthe press. Liquid nitrogen can be added periodically to the telasubmucosa specimens to keep them frozen.

Other methods for comminuting tela submucosa specimens can be utilizedto produce a tela submucosa powder usable in accordance with the presentinvention. For example, tela submucosa specimens can be freeze-dried andthen ground using a manual arbor press or other grinding means.Alternatively, tela submucosa can be processed in a high shear blenderto produce, upon dewatering and drying, a tela submucosa powder.

Further grinding of the tela submucosa powder using a prechilled mortarand pestle can be used to produce consistent, more finely dividedproduct. Again, liquid nitrogen is used as needed to maintain solidfrozen particles during final grinding. The powder can be easilyhydrated using, for example, buffered saline to produce a fluidizedtissue graft material of this invention at the desired viscosity.

To prepare another preferred fluidized material, a tela submucosa powdercan be sifted through a wire mesh, collected, and subjected toproteolytic digestion to form a substantially homogeneous solution. Forexample, the powder can be digested with 1 mg/ml of pepsin (SigmaChemical Co., St. Louis Mo.) and 0.1 M acetic acid, adjusted to pH 2.5with HCl, over a 48 hour period at room temperature. After thistreatment, the reaction medium can be neutralized with sodium hydroxideto inactivate the peptic activity. The solubilized submucosa can then beconcentrated by salt precipitation of the solution and separated forfurther purification and/or freeze drying to form a protease-solubilizedintestinal submucosa in powder form.

Fluidized tela submucosa compositions of this invention find wideapplication in tissue replacement, augmentation, and/or repair. Thefluidized submucosal compositions can be used to induce regrowth ofnatural connective tissue or bone in an area of an existent defect. Byinjecting an effective amount of a fluidized submucosa composition intothe locale of a tissue defect or a wound in need of healing, one canreadily take advantage of the biotropic properties of the telasubmucosa.

It is also possible to form large surface area constructs by combiningtwo or more tela submucosa segments of the invention, for instance usingtechniques as described in U.S. Pat. No. 2,127,903 and/or InternationalPublication No. WO 96/32146, dated 17 Oct. 1996, publishingInternational Application No. PCT/US96/04271, filed 5 Apr. 1996. Thus, aplurality of tela submucosa strips can be fused to one another, forexample by compressing overlapping areas of the strips under dehydratingconditions, to form an overall planar construct having a surface areagreater than that of any one planar surface of the individual stripsused to form the construct.

Tela submucosa of the invention can also be employed to prepare tissuegraft constructs useful in orthopedic soft tissue applications, forexample in tendon or ligament repair, employing techniques in the artwhich have been applied to other naturally-derived or synthetic graftmaterials. For instance, repair techniques as generally described inU.S. Pat. Nos. 2,127,903 and 5,281,422 can be undertaken using telasubmucosa of the present invention.

For tendon and ligament replacement applications, a segment of the telasubmucosa can be preconditioned by longitudinal stretching to anelongated length. For example, a tela submucosa segment can beconditioned by the prolonged application of a load on the longitudinalaxis of the segment (e.g. by suspending a weight from the segment) for aperiod of time sufficient to allow about 10% to about 20% elongation ofthe tissue segment. The graft material can also be preconditioned bystretching in the lateral dimension. The tela submucosa segment can thenbe configured, alone or in combination with other segments, to a varietyof shapes to serve as a ligament or tendon replacement, or to substitutefor or patch a broken or severed tendon or ligament.

For such connective tissue grafting applications, the segment isdesirably configured to have a layered or multilayered configuration,with at least the opposite end portions and/or opposite lateral portionsbeing formed to have multiple layers of the graft material to providereinforcement for attachment to physiological structures such as bone,tendon, ligament, cartilage and muscle. In a ligament replacementapplication, opposite ends will be attached to first and second bones,respectively, the bones typically being articulated as in the case of aknee joint. In a tendon replacement application, a first end of thegraft construct will be attached to a bone, and a second end will beattached to a muscle.

As indicated above, in connective tissue applications, it will beadvantageous to form, manipulate or shape the end portions of the graftconstruct to be attached, for example, to a bone structure, in a mannerthat will reduce the possibility of graft tearing at the point ofattachment. For these purposes, the tela submucosa graft material can befolded or partially everted to provide multiple layers for gripping, forexample, with spiked washers or staples. Alternatively, a tela submucosasegment can be folded back on itself to join the end portions to providea first connective portion to be attached, for example, to a first boneand a bend in the intermediate portion to provide a second connectiveportion to be attached to a second bone articulated with respect to thefirst bone.

For example, one of the end portions of the tela submucosa graft can beadapted to be pulled through a tunnel in, for example, the femur andattached thereto, while the other of the end portions may be adapted tobe pulled through a tunnel in the tibia and attached thereto to providea substitute for the natural cruciate ligament, the segment beingadapted to be placed under tension between the tunnels to provide aligament function, i.e., a tensioning and position function provided bya normal ligament.

Because grafts used in orthopedic applications are typically placedunder tension in their surgical installation, it is preferable tocombine two or even more tissue segments to provide a multi-ply(multi-layered) graft construct. It is another object of the presentinvention, therefore, to provide such grafts in which two or moresubmucosa segments are arranged to have their end portions joinedtogether with the joined end portions and/or lateral portions adapted tobe attached to a bone, tendon, ligament or other physiologicalstructure. One method for providing a double segment can be to pull onetubular segment internally within another segment to provide adouble-walled tube, the joined ends of which can be attached, forexample, to a bone, tendon or ligament. These doubled segments willprovide enhanced tensile strength and retela submucosatance tostretching under tension. In other forms, multiple tela submucosasegments or strips can be arranged in a braided configuration, forexample a diamond or sashcord braided configuration, or in a meshconfiguration including multiple loops intercoupled to neighboringloops, which usefully serve in ligament or tendon repair.

Tela submucosa of the present invention can also be used to provide anorthopedic graft for use as connective tissue to hold fractured bonepieces together and in proper orientation in the body, the tissuesegment being formed to serve as a fracture wrap about segments offractured bone and to be attached to the bone.

In still further orthopedic applications, tela submucosa of theinvention can be used to repair bone tissue, for instance using thegeneral techniques described in U.S. Pat. No. 5,641,518. Thus, a powderform of the tela submucosa can be implanted into a damaged or diseasedbone region for repair. The tela submucosa powder can be used alone, orin combination with one or more additional bioactive agents such asphysiologically compatible minerals, growth factors, antibiotics,chemotherapeutic agents, antigen, antibodies, enzymes and hormones.Preferably, the powder-form implant will be compressed into apredetermined, three-dimensional shape, which will be implanted into thebone region and will substantially retain its shape during replacementof the graft with endogenous tissues.

Tela submucosa of the invention can also be used as a cell growthsubstrate, illustratively in sheet, paste or gel form in combinationwith nutrients which support the growth of the subject cells, e.g.eukaryotic cells such as endothelial, fibroblastic, fetal skin,osteosarcoma, and adenocarcinoma cells (see, e.g. InternationalPublication No. WO 96/24661 dated 15 Aug. 1996, publishing InternationalApplication No. PCT/US96/01842 filed 9 Feb. 1996. In preferred forms,the tela submucosa substrate composition will support the proliferationand/or differentiation of mammalian cells, including human cells.

The inventive tela submucosa can also serve as a collagenous matrix incompositions for producing transformed cells, (see, e.g., InternationalPublication No. WO 96/25179 dated 22 Aug. 1996, publishing InternationalApplication No. PCT/US96/02136 filed 16 Feb. 1996; and InternationalPublication No. WO 95/22611 dated 24 Aug. 1995, publishing InternationalApplication No. PCT/US95/02251 filed 21 Feb. 1995). Such compositionsfor cell transformation will generally include purified tela submucosaof the present invention, for example in fluidized or paste form, incombination with a recombinant vector (e.g. a plasmid) containing anucleic acid sequence with which in vitro or in vivo target cells are tobe genetically transformed. The cells targeted for transformation caninclude, for example, bone progenitor cells.

Tela submucosa of the invention can also be used in body wall repair,including for example in the repair of abdominal wall defects such ashernias, using techniques analogous to those described in Ann. Plast.Surg., 1995, 35:3740380; and J. Surg. Res., 1996, 60:107-114. In suchapplications, preferred tela submucosa tissue grafts of the inventionpromote favorable organization, vascularity and consistency in theremodeled tissue. In dermatological applications, tela submucosa of theinvention can be used in the repair of partial or full thickness woundsand in dermal augmentation using general grafting techniques which areknown to the art and literature (see, e.g. Annals of Plastic Surgery1995, 35:381-388). In addition, in the area of burn treatment, it isgenerally known to provide a dermal substitute onto which culturedepidermal grafts (preferably cultured epidermal autografts, or CEA's)are transplanted. Such cultured grafts have typically involvedtransplanting keratinocytes and/or fibroblasts onto the dermalsubstitute. In accordance with the present invention, the purified telasubmucosa can be used as the dermal substitute, for example in sheetform, and the CEA accordingly transplanted onto the tela submucosa. Inone mode of practicing this aspect of the invention, keratinocytes canbe transplanted, for example by seeding or transferring a keratinocytesheet, onto the mucosal side of the tela submucosa. Fibroblasts can betransplanted also on the mucosal and/or on the opposite (abluminal) sideof the tela submucosa.

Tela submucosa of the invention can also be used in tissue grafting inurogenital applications. For instance, the tela submucosa can be used inurinary bladder repair to provide a scaffold for bladder regeneration,using techniques corresponding to those generally described in U.S. Pat.No. 5,645,860; Urology, 1995, 46:396-400; and J. Urology, 1996,155:2098. In fluidized form, the inventive tela submucosa can also finduse in an endoscopic injection procedure to correct vesicureteralreflux. In such applications, a submucosal injection can be made, forinstance in the area under the ureteral orifice of a patient, to inducesmooth muscle growth and collagen formation at the injection site.

In other areas, tissue graft constructs formed with tela submucosa ofthe present invention can be used in neurologic applications, forexample in techniques requiring a dural substitute to repair defects dueto trauma, tumor resection, or decompressive procedures.

In order to promote a further understanding of the present invention andits features and advantages, the following specific Examples areprovided. It will be understood that these specific Examples areillustrative, and not limiting, of the present invention.

EXAMPLE 1

Thirty feet of whole intestine from a mature adult hog is rinsed withwater. This material is then treated in a 0.2 percent by volumeperacetic acid in a 5 percent by volume aqueous ethanol solution for aperiod of two hours with agitation. The tela submucosa layer is thendelaminated in a disinfected casing machine from the whole intestine.The delaminated tela submucosa is rinsed four (4) times with sterilewater and tested for impurities or contaminants such as endotoxins,microbial organisms, and pyrogens. The resultant tissue was found tohave essentially zero bioburden level. The tela submucosa layerseparated easily and consistently from the whole intestine and was foundto have minimal tissue debris on its surface.

EXAMPLE 2

A ten foot section of porcine whole intestine is washed with water.After rinsing, this section of tela submucosa intestinal collagen sourcematerial is treated for about two and a half hours in 0.2 percentperacetic acid by volume in a 5 percent by volume aqueous ethanolsolution with agitation. Following the treatment with peracetic acid,the tela submucosa layer is delaminated from the whole intestine. Theresultant tela submucosa is then rinsed four (4) times with sterilewater. The bioburden was found to be essentially zero.

EXAMPLE 3

A small section of the tela submucosa intestinal collagen material wassubcutaneously implanted in a rat. Within 72 hours, significantangiogenesis was observed.

EXAMPLE 4

Two sections of small intestine are processed by differing methods. Thefirst section is rinsed in tap water, disinfected for 2 hours in a 5% byvolume aqueous ethanol solution comprising 0.2% by volume peraceticacid, pH approximately 2.6, delaminated to the tela submucosa, rinsed inpurified water, divided into two samples and rapidly frozen. The secondsection is rinsed in tap water, delaminated to the tela submucosa,rinsed in purified water, placed in a 10% neomycin sulfate solution for20 minutes (as described in U.S. Pat. No. 4,902,508), rinsed in purifiedwater, divided into two samples and rapidly frozen. The fourabove-prepared samples are tested for bioburden and endotoxin levels.The first two samples each have bioburdens of less than 0.1 CFU/g andendotoxin levels of less than 0.1 EU/g. The second two samples haverespective bioburdens of 1.7 CFU/g and 2.7 CFU/g and respectiveendotoxin levels of 23.9 EU/g and 15.7 EU/g.

EXAMPLE 5

Three sections of small intestine are processed by differing methods.The first is rinsed in tap water, disinfected for 2 hours in a 5% byvolume aqueous ethanol solution comprising 0.2% by volume peraceticacid, pH about 2.6, delaminated to the tela submucosa, rinsed inpurified water, and rapidly frozen. The second is rinsed in tap water,delaminated to tela submucosa, rinsed in purified water, disinfectedaccording to the methods of Example 1 in U.S. Pat. No. 5,460,962(treatment for 40 hours in a 0.1% by volume aqueous solution ofperacetic acid, buffered to pH 7.2), and rapidly frozen. The third isrinsed in tap water, delaminated to the tela submucosa, rinsed inpurified water, disinfected according to the methods of Example 2 inU.S. Pat. No. 5,460,962 (treatment in 0.1% by volume peracetic acid inhigh salt solution, buffered to pH 7.2), and rapidly frozen. All threesamples were tested for endotoxins. The endotoxin levels were <0.14 EU/gfor the first sample, >24 EU/g for the second sample, and >28 EU/g forthe third sample.

EXAMPLE 6

Two sections of porcine small intestine were infected with 7×10⁶ plaqueforming units (PFU) of virus. Both were exposed to a 0.18% peraceticacid, 4.8% aqueous ethanol solution at a nine-to-one weight ratio ofsolution to material. A first sample was immersed in this solution for 5minutes; the second was immersed for 2 hours. The material processed for5 minutes exhibited 400 PFU per gram of material. The material processedfor 2 hours exhibited zero PFU per gram of material.

EXAMPLE 7

Purified tela submucosa, prepared as described herein, was tested todetermine its nucleic acid content. Four samples of material weighing 5mg each were subjected to DNA/RNA extraction as detailed in the DNA/RNAIsolation Kit by Amersham Lifescience Inc., Arlington Heights, Ill.Nucleic acid quantitation was performed by spectrophotometricdetermination of solution optical densities at 260 nm and 280 nm. Theaverage nucleic acid content was 1.9±0.2 μg per milligram of material.

Small intestinal submucosa, prepared as described by U.S. Pat. No.4,902,508, was tested to determine its nucleic acid content. Foursamples of material weighing 5 mg each were subjected to DNA/RNAextraction as detailed in the DNA/RNA Isolation Kit by Amersham. Nucleicacid quantitation was performed by spectrophotometric determination ofsolution optical densities at 260 nm and 280 nm. The average nucleicacid content was 2.4±0.2 μg per milligram of material.

EXAMPLE 8

Sections of tela submucosa prepared according to the methods describedherein were sent to an independent testing laboratory (NamSA, Inc.,Northwood, Ohio) for biocompatibility testing as described in thestandard ISO 10993. The samples were tested for USP Acute SystemicToxicity, USP Intracutaneous Toxicity, Cytotoxicity, LAL Endotoxin,material-mediated Pyrogenicity, Direct Contact Hemolysis, and PrimarySkin Irritation. The samples passed all tests, indicating that thematerial is biocompatible.

It will be appreciated that variations of the above-described processingprocedures are intended to be within the scope of this invention. Forexample, the source tissue for the tela submucosa, e.g., stomach, wholeintestine, cow uterus and the like, can be partially delaminated,treated with a disinfecting or sterilizing agent followed by completedelamination of the tela submucosa. Illustratively, attached mesenterylayers, and/or serosa layers of whole intestine can be advantageouslyremoved prior to treatment with the disinfecting agent, followed bydelamination of remaining attached tissues from the tela submucosa.These steps may or may not be followed by additional disinfection steps,e.g., enzymatic purification and/or nucleic acid removal. Alternatively,the tela submucosa source can be minimally treated with a disinfectingor other such agent, the tela submucosa delaminated from the tunicamuscularis and tunica mucosa, followed by a complete disinfectiontreatment to attain the desired contaminant level(s). All suchvariations and modifications are contemplated to be a part of theprocess described herein and to be within the scope of the invention.

In addition, it will be appreciated that the publications cited hereinare indicative of the skill possessed by those practiced in the relevantfield, and each such publication is hereby incorporated by reference inits entirety as if individually incorporated by reference and fully setforth.

1.-64. (canceled)
 65. A collagen-containing matrix obtained from asubmucosa tissue source, comprising: a collagen-containing matrixobtained from a submucosa tissue source from urinary tract tissue, thecollagen-containing matrix including submucosa tissue and lamina propriatissue from the tissue source, wherein the submucosa tissue has beenseparated from tunica muscularis tissue that was present in thesubmucosa tissue source; wherein said collagen-containing matrixexhibits a bioburden of less than 0.5 colony forming units per gram;wherein said collagen-containing matrix retains growth factors from saidtissue source, and said growth factors include basic fibroblast growthfactor; and wherein said collagen-containing matrix exhibits a capacityto induce angiogenesis.
 66. A method for treating a patient, comprising:providing a collagen-containing matrix obtained from a submucosa tissuesource, the collagen-containing matrix comprising: a collagen-containingmatrix obtained from a submucosa tissue source from urinary tracttissue, the collagen-containing matrix including submucosa tissue andlamina propria tissue from the tissue source, wherein the submucosatissue has been separated from tunica muscularis tissue that was presentin the submucosa tissue source; wherein said collagen-containing matrixexhibits a bioburden of less than 0.5 colony forming units per gram;wherein said collagen-containing matrix retains growth factors from saidtissue source, and said growth factors include basic fibroblast growthfactor; and wherein said collagen-containing matrix exhibits a capacityto induce angiogenesis; and treating a partial or full thickness dermalwound of the patient with the collagen-containing matrix.
 67. The methodof claim 66, wherein the tissue source is porcine.
 68. The method ofclaim 66, wherein the collagen-containing matrix has a nucleic acidcontent level of less than 2 micrograms per milligram; a virus level ofless than 5 plaque forming unit per gram; and a fungus level of lessthan 0.5 colony forming units per gram.
 69. The method of claim 66,wherein the collagen-containing matrix is provided in a sterilecondition.
 70. The method of claim 69, wherein the collagen-containingmatrix has been sterilized through contact with an aqueous solutioncontaining a peracid.
 71. The method of claim 70, wherein the peracid isperacetic acid.
 72. The method of claim 66, wherein thecollagen-containing matrix has not been treated with a crosslinkingagent.
 73. The method of claim 66, wherein the collagen-containingmatrix is provided in a fluidized form.
 74. The method of claim 66,wherein the collagen-containing matrix is provided in the form of a gel.75. The method of claim 66, wherein the collagen-containing matrixcomprises a first collagenous matrix material layer and a secondcollagenous matrix material layer providing a multilayer construct. 76.The method of claim 75, wherein said first collagenous matrix materiallayer is fused to said second collagenous matrix material layer.
 77. Themethod of claim 76, wherein said first collagenous matrix material layerhas been fused to said second collagenous matrix material layer bycompressing overlapping areas of the first material layer and secondmaterial layer under dehydrating conditions.
 78. The method of claim 66,wherein the collagen-containing matrix has a bioburden level ofsubstantially zero.
 79. The method of claim 66, wherein thecollagen-containing matrix is provided in the form of a powder.
 80. Themethod of claim 79, wherein the powder is combined with an aqueouscarrier so as to form an injectable fluid composition.
 81. The method ofclaim 66, wherein the collagen-containing matrix is in the form of asheet.